The mosquito- and tick-borne viruses within the Flaviviridae family comprise important emerging and re-emerging pathogens Japanese encephalitis (JEV), tick-borne encephalitis (TBEV), West Nile (WNV), St. Louise encephalitis (SLEV) and Zika (ZIKV) viruses that have caused outbreaks of severe and often fatal diseases in humans in many regions of the world. During the past decades, both mosquito- and tick-borne flaviviruses expanded their geographic range and emerged in new areas where their spread resulted in severe impact on wildlife. The Neurotropic Flaviviruses Section (NFS) is currently engaged in the development of vaccines against diseases caused by neurotropic flaviviruses from three major groups: JEV serocomplex, TBEV serocomplex and ZIKV. JEV is transmitted in Southeast Asia and results in an estimated 60,000 annual cases of which about 30% are fatal. SLEV is endemic in Central and South America, and more than 10,000 cases of neuroinvasive SLEV disease have been reported in the USA. Currently, WNV is the major source of viral encephalitis in the North America. During the 1999-2016 epidemics, nearly 3 million people were infected with WNV in the US, with about 21,078 CDC-reported cases of neuroinvasive disease and 1985 deaths, majority of which occurred in the elderly. Over the last decade, ZIKV had emerged explosively in Latin America and is associated with catastrophic fetal abnormalities including microcephaly and spontaneous abortion. Unlike other members of the Flaviviridae family, ZIKV was able to establish persistent infection in humans and can be transmitted sexually, posing new challenges for controlling virus outbreaks. Live-attenuated virus vaccines are known to have several favorable advantages compared to other vaccine formulations and elicit a durable protective immunity after a single dose. Therefore, development of safe and efficacious vaccines is of significant benefit for public health in prevention of neurotropic virus-associated illness in populations living in endemic areas. Successful attenuation of a neurotropic virus pathogenesis depends on the prevention of virus entry into the central nervous system (CNS) and restriction of its replication in the neurons. Thus, to limit virus access into CNS, the attenuated vaccine candidates against neurotropic JEV, WNV, SLEV and TBEV are being developed in the NFS using the strategies based on chimerization of a neurovirulent virus with a non-neuroinvasive dengue type 4 flavivirus (DEN4) or naturally attenuated tick-borne Langat virus (LGT) and introduction of attenuating mutations. To restrict viral replication in the neurons, we have developed and utilized an effective strategy for selective control of virus neurotropism by targeting of viral genome for cellular microRNAs (miRNAs) expressed in the brain. The most attenuated vaccine candidates were then evaluated for safety, immunogenicity, their ability to protect mice and monkeys against challenge with wild-type virulent virus. In addition, a miRNA targeting approach was adapted to design environmentally-safe vaccine viruses restricted in their ability to infect and be transmitted by competent arthropod vectors. JE, SLE and WN vaccines: Our first generation of WNV vaccine is a chimeric virus in which the prM and E genes of the DEN4 virus have been replaced by those of WNV. A vaccine virus was unable to replicate in the CNS of non-human primates - infectious virus and viral antigens were not detected in the brain or spinal cord. However, in the CNS of WNV-infected monkeys, anatomical mapping revealed that all structures identified as containing WNV-labeled neurons belong to the pathways of motor control system (Maximova et al., 2016). WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons. As described in the publication by Pierce et al. (J Infect Dis 2017; 215(1):52-55), a WNV vaccine was shown to be safe, well-tolerated, and effective in healthy elderly volunteers, adults aged 50-65 years. Immunization with a single vaccine dose produces prominent level of neutralizing antibody against wild-type WNV strains, inducing a 95% rate of seroconversion in the volunteers. These results suggest that a single dose of vaccine may be sufficient to induce a protective antibody response in the vulnerable elderly population. In FY2016-17, the next generation of vaccine candidates was engineered by introducing multiple targets for the CNS-specific miRNAs into WN/DEN4, JE/DEN4 and SLE/DEN4 genomes. A single dose of bivalent vaccine formulations (WN/DEN4 and SLE/DEN4 miRNA-targeted viruses) induced strong neutralizing WNV- and SLEV-specific antibody responses and protected mice against parental WNV or SLEV challenge. Currently, we are working to identify the most attenuated JEV vaccine candidate for neuropathogenesis in mice that will be used as a third component of the combined trivalent vaccine against WNV, SLEV and JEV. TBEV vaccines: The TBEV vaccine candidates are chimeric viruses carrying the prM and E structural protein genes of Far Eastern TBEV and remaining sequences derived from either (1) a non-neuroinvasive DEN4 (TBEV/DEN4), (2) a naturally attenuated, tick-borne LGT strain E5 (TBEV/E5) or (3) a most immunogenic LGT strain T1674 (TBEV/LGT). A large set of modified TBEV/DEN4 and TBEV/LGT was generated by introducing the cassettes of brain-specific miRNA-targets into three distant regions of chimeric DEN4- or LGT-based genome, etc., in the duplicated capsid gene region, the duplicated E gene region, and the 3NCR. In FY2017, we identified most promising DEN4- and LGT-based vaccine candidates that (1) were completely restricted for replication in the brain of newborn mice, (2) had significantly limited neuroinvasiveness in immunodeficient mice and (3) were genetically stable during prolonged replication in the periphery. In comparative studies DEN4- and LGT-based viruses in rhesus monkeys, we demonstrated that both vaccine candidates are safe and able to induce protective immunity to challenge with Far Eastern subtype of TBEV (unmodified TBEV/LGT virus). The extensive studies in mice revealed that LGT-based vaccine candidates were able to provide complete protection against severe challenge with heterotypic European TBEV (strain Hypr). Recently (Tsetsarkin et al., Sci Rep 2016), we showed that insertion of target sequences for tick-specific miRNAs (mir-1, mir-275 and mir-279) individually or in combination into tick-borne LGT genome caused selective attenuation of virus replication in tick-derived cells and live Ixodes ticks. Subsequently, we are planning to insert the target sequences for these miRNAs into TBEV/LGT genomes and design the environmentally-safe vaccines by restricting their replication in the ticks and ability to be introduced into nature. ZIKV cDNA clone: In FY2017, we developed and characterized a full-length infectious cDNA clone of ZIKV isolated during 2015 epidemic in Brazil (Tsetsarkin et al., mBio 2016). A Vero cell-adapted cDNA clone of ZIKV, as well as viruses expressing reporter genes (GFP and Luciferase) were also generated that can be used as a convenient genetic platform for studies of virus evolution, virus-host interactions and vaccine development. Viruses replicated efficiently in Vero cells, reaching titer of 7-8 log10(pfu/ml), and were genetically stable during serial passaging in Vero cells. Using ZIKV cDNA clone, we created a panel of viruses expressing complementary targets for reproductive organ tissue- and CNS-specific cellular microRNAs. Analysis of the effect of miRNA targets on ZIKV growth in cells derived from brain, placenta, testis and mosquitoes as well as on ZIKV pathogenesis in mice is ongoing.